Exposure to "atypical" or "environmental" mycobacteria [EM] is believed to be the primary mechanism thought to be responsible for the reduced efficacy of the BCG vaccine against tuberculosis in many parts of the world. To date however, the exact mechanism by which EM interfere with the development of protective immunity in response to vaccination still remains unknown. It has been hypothesized that constant exposure to cross-reactive antigens can sequester specific cells at alternative sites in the body, or that the immune response generated against environmental mycobacteria is strong enough to control BCG before its protective effect can be acquired. The studies documented in the literature however have used several different exposure protocols making interpretation of the results very difficult. This application seeks to carry out an analytical study using standardized exposure protocols. We propose to administer environmental mycobacteria either before BCG vaccination (to determine whether environmental exposure interferes with the generation of immunity against the vaccine) or following BCG vaccination (to determine whether environmental exposure influences the long-term efficacy of BCG). Throughout the study we will isolate immune cells from the draining lymph nodes and lungs and determine whether cells become sequestered at the site of environmental exposure, become highly activated in response to mycobacterial antigens, or become deleted from the lymphocyte pool. Using these methods we aim to determine the basis of the microbial interactions between EM and BCG that underlies the failure of the vaccine to protect against infection with M. tuberculosis. In addition, we propose to carry out a preliminary proteomics study to identify key antigens from M. tuberculosis that are absent from EM that could be incorporated into a sub-unit vaccine.